Transcriptional Induction of NF-kB-Inducing Kinase by E2F4/5 Facilitates Collective Invasion of Glioma Cells

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway increases the invasiveness of glioma cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest expression observed in the most invasive cells. Glioma cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote glioma cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting glioma pathogenesis.


Introduction
While glioblastoma multiforme (GBM) tumors rarely metastasize outside of the central nervous system (CNS), their aggressive growth and persistent invasiveness into healthy brain tissue are major factors underlying resistance to conventional treatment methods such as surgery, irradiation, and chemotherapy 1,2 . In addition to single-cell invasion, multicellular, connected networks of glioma cells have been observed in human tumors and mouse models [3][4][5] . This collective invasion, de ned by the coordinated movement of cells into surrounding tissue while maintaining cell-cell junctions, is a process that occurs in epithelial regeneration and during the development and remodeling of large tissue structures, including angiogenesis and neural crest cell streaming. Collective invasion has also been identi ed as a signi cant mode of invasion in glioma 6,7 . During collective invasion, cells have been found to acquire leader-follower phenotypes, in which both leader (pioneer) and follower cells assume different metabolic activities and work in concert to promote tumor cell dispersion. Leader cells direct the leading edge of the tumor, migrating through the microenvironment, paving the path of invasion, and transmitting information to follower cells 8,9 . However, there is an increasing body of evidence that tumor cells in collective invasion may alternate between leader and follower phenotypes, warranting further studies of this process.
Activation of NF-κB signaling pathways has been well documented in a variety of malignancies, including GBM 10 . We previously demonstrated that activation of the noncanonical NF-κB pathway through TNFweak-like factor (TWEAK) is a potent inducer of GBM invasion 11 . Speci cally, NF-κB-inducing kinase (NIK; encoded by MAP3K14), which is an essential upstream kinase for noncanonical NF-κB activation, increases matrix metalloproteinase 1 (MT1-MMP) activity to promote invadopodia formation during glioma invasion 12 . NIK is generally described as being regulated at the posttranslational level, whereby constitutive proteosome-dependent NIK protein turnover is attenuated in a signal-dependent manner, resulting in the accumulation of catalytically active NIK. In the current study, we show that the dynamic, signal-dependent transcriptional upregulation of NIK is pronounced in cells at the leading edge of gliomaspheres, enhancing collective invasion of neighboring cells into the surrounding matrix.

Induction of NIK Transcription Directly Correlates with Invasion
Consistent with our previous ndings 11 , we observed that the invasiveness of the human-derived glioblastoma (GBM) cell lines BT25, BT114, and BT116 was signi cantly induced by treatment with TWEAK, whereas treatment with TNFα did not stimulate invasion ( Figure 1A). Transcriptome analysis of BT25 cells treated with TWEAK or TNFα, which preferentially activate the canonical or noncanonical NF-κB pathways, respectively 13,14 , revealed that the expression of NIK (MAP3K14) directly correlated with glioma cell invasion and was highly induced in response to TWEAK treatment but not TNFα treatment ( Figure 1B, C). Moreover, we observed TWEAK-speci c upregulation of tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1), a signaling adapter that interacts with and stabilizes NIK for activation of the noncanonical NF-κB pathway 15 . Elevated levels of integrin β3 (ITGB3), integrin subunit alpha 11 (ITGA11), matrix metalloproteinase 9 (MMP9), and Fms-related tyrosine kinase (FLT1) were also observed, consistent with increased invasive and migratory potential 16 . In addition to the induction of ITGB3, we observed that TWEAK treatment also elevated the expression of ITGA11, which has previously been described in glioma invasion 17 . Furthermore, only TWEAK-treated glioma cells exhibited increased expression of integrins, as well as MMP9, all of which are associated cancer markers 18-21 . Ingenuity Pathway Analysis (IPA) of groups of genes belonging to speci c diseases and functions revealed that TWEAK treatment elevated the overall expression of cancer pathways, including tumor formation, invasion, and metastasis ( Figure 1D). Additionally, RNA-seq analysis demonstrated that relative to other MAP3 kinases and upstream regulatory kinases in the NF-κB signaling pathways, TWEAK treatment singularly induced NIK/MAP3K14 gene expression ( Figure 1E). For example, while qPCR analysis showed high expression of MAP3K14 as well as MAP3K8 in TWEAK-treated cells, MAP3K8 was also induced by both TNF and TWEAK. Moreover, analysis of cells actively undergoing invasion in collagen matrices revealed that NIK/MAP3K14 was the most highly upregulated gene compared to other kinases ( Figure   1F). We also observed TWEAK-induction of NIK mRNA and invasion in other GBM cell lines, as well as mouse embryonic broblasts (MEFs) (Supp. Figure 1A, B, C).
After observing elevated NIK transcription and invasion upon TWEAK treatment, we investigated potential paracrine effects among glioma cells during invasion. We found that BT116 cells underwent increased invasion when treated with conditioned media (Control CM) from highly invasive BT25 cells compared with unconditioned media. This increased invasion was dependent on NIK, as it was not observed when BT116 cells were cultured with conditioned media from NIK knockout BT25 cells (NIK KO CM), while conditioned media from NIK KO cells rescued with ectopic expression of murine NIK (NIK KO-mNIK CM) restored the increased invasion (Supp. Figure 1D-E). Consistent with the results from media treatments, direct coculture of BT25 and BT114 cells increased the cell invasion/migration of the BT114 cells compared to cells cultured alone (Supp. Figure 1F). These results demonstrate that transcriptional induction of NIK is associated with elevated glioma invasiveness that is propagated by NIK-dependent paracrine signaling.

NIK Expression is Upregulated in Highly Invasive Glioma Cells and Promotes Collective Invasion
To monitor the induction of NIK transcription during invasion in vivo, we generated BT116 glioma cells stably expressing red uorescent protein (RFP) under the promoter of NIK (pNIK-RFP). Analysis of invading BT116 pNIK-RFP cells revealed a general induction of NIK expression in invading cells (red signal from monolayer pseudocolored white) (Figure 2A), with the farthest invading cells exhibiting the highest RFP intensity or highest expression of NIK ( Figure 2B). Treatment of BT116 pNIK-RFP cells with TWEAK further increased NIK expression (RFP signal-red/yellow), with RFP-positive cells invading farther under either untreated (NT) or TWEAK-treated conditions ( Figure 2C).
To evaluate collective invasion and leader-follower phenotypes in a 3-dimensional view, we performed invasion assays of glioma tumor spheres, which better mimic cell-cell and cell-matrix interactions. Imaging of collagen-embedded BT116 pNIK-RFP spheres revealed an increase in pNIK-RFP expression upon TWEAK treatment ( Figure 2D), with the highest pNIK-RFP expression observed among the most invasive cells at the leading edge of the sphere (Supp. Fig 2A, Figure 2D, E). Consistent with single-cell invasion assays, the induction of NIK expression directly correlated with the migration and dispersion of cells from glioma spheres, which was enhanced upon TWEAK treatment ( Figure 2F) compared to the invasion of similar-sized spheres from the untreated group ( Figure 2G). Cells with a high pNIK-RFP signal were observed among cells undergoing collective invasion at the sphere peripheries ( Figure 2H). These results demonstrate that cells with the highest TWEAK-induced NIK expression directly correlated with the most invasive glioma cells, facilitating collective invasion, consistent with increased invasion and metastasis gene signatures.

Inhibition of NIK Activity Reduces Glioma Cell Invasion
Next, we evaluated whether NIK catalytic activity was required to promote GBM invasion. Treatment of cells with mangiferin, a natural inhibitor of NIK [22][23][24] , signi cantly attenuated TWEAK-stimulated invasion to levels comparable with untreated or TNF-treated conditions ( Figure 3A, B). Mangiferin was veri ed to inhibit activation of the noncanonical NF-κB pathway by TWEAK treatment in glioma cells, as seen with a reduction in p100-p52 processing and RelB ( Figure 3C). Mangiferin also inhibited TWEAK-induced NIK transcription (Supp. Fig. 2B). Although mangiferin inhibited NIK and the noncanonical NF-κB pathway, it did not affect cell proliferation (Supp. Figure 2C). These data suggest therapeutic potential of inhibiting NIK to attenuate glioma invasion.

E2F4 and E2F5 Regulate NIK Gene Expression
The mechanisms underlying the regulation of NIK gene expression are not fully understood. A recent study identi ed MAP3K14 (NIK) as a signi cantly upregulated gene in human osteosarcoma cells with E2F activation 25 , and analysis of the NIK promoter revealed the presence of E2F binding sites (Supp. Fig.   3D). Thus, we next investigated whether E2F transcription factors played a role in early NIK gene expression in response to TWEAK treatment in glioma cells. We observed that TWEAK treatment increased nuclear E2F4 and E2F5 protein levels in glioma cells, with E2F4 having greater nuclear translocation in BT25 cells and E2F5 in BT114 cells ( Figure 4A). We also found that overexpression of E2F5, but not E2F1, increased NIK transcript levels (Supp. Figure 3A). E2F4 and E2F5 single and double knockout cell lines generated by CRISPR-Cas9 gene editing exhibited a signi cant reduction in NIK protein levels when treated with TWEAK and MG132 to inhibit proteosome-dependent degradation (Supp. Figure   3B, Figure 4B). Moreover, E2F4/5 double knockout cells (E2F DKO) exhibited reduced p52/RelB nuclear translocation, demonstrating impaired activation of NIK-driven noncanonical NF-κB signaling (Supp. Figure 3C).
Next, we investigated whether E2F proteins directly regulate NIK transcription. Chromatin immunoprecipitation (ChIP) analyses demonstrated that antibodies speci c to E2F4 and E2F5, but not IgG, were bound to certain regions of the NIK promoter ( Figure 4C). Additionally, qPCR analysis of E2F-DKO cells showed signi cantly attenuated induction of NIK mRNA expression even with TWEAK treatment ( Figure 4D). Reduced NIK expression in E2F DKO cells proved to have functional consequences, as these cells were poorly invasive, even after TWEAK treatment ( Figure 4E), which was restored with ectopic expression of NIK (NIK OE) in E2F DKO cells ( Figure 4F). Overall, these data establish a novel role for E2F regulation of NIK transcription in a stimulated state and thus affect the cell's overall ability to invade (Supp. Fig. 3F).

Discussion
While posttranscriptional regulation of NIK protein stability is important for controlling the activation of NF-κB signaling, in this study, we report that signal-speci c transcriptional upregulation of NIK, but not other related kinases, is strongly associated with enhanced collective invasion of glioma cells (Figure 1). RNA-seq and qPCR analyses also veri ed that TWEAK but not TNFα treatment elevated NIK transcription, which was also observed in actively invading glioma cells. Using a NIK promoter-controlled reporter construct as a readout for NIK gene expression levels, we demonstrate that TWEAK-induced NIK transcription directly correlates with cell invasion and the acquisition of leader-follower cells seen in collective invasion ( Figure 2H). Indeed, we observed that the farthest invading cells expressed the highest levels of NIK transcription ( Figure 2). Our data showing that conditioned media from NIK KO glioma cells was unable to stimulate invasion, while conditioned media from NIK KO-mNIK cells rescued invasion, suggests that NIK-dependent paracrine signaling propagates a collective leader-follower cell phenotype during cell invasion. Furthermore, although prior studies have shown that E2F regulation of cIAPs and E2F1 directly promotes MAP3K14 gene expression in osteosarcoma cells [25][26][27] , we report for the rst time that the E2F transcription factors E2F4 and E2F5 play a role in regulating NIK transcription and expression. Overall, these data reveal critical roles for the regulation of NIK at the transcriptional level in propagating glioma cell invasion.
Utilizing speci c cytokine treatments for preferential activation of the canonical or noncanonical NF-κB pathway by TWEAK or TNFα, respectively, we demonstrated that noncanonical NF-κB pathway activation increased invasion. Although we demonstrate that NIK promotes invasion in a noncanonical NF-κBdependent manner, this does not exclude possible involvement of the canonical NF-κB pathway in regulating cancer invasion through independent or complementary mechanisms. Furthermore, we have previously demonstrated that NIK promotes mitochondrial ssion and tra cking to the periphery of glioma cells during cell migration in a manner that is independent of IKKα/β and downstream NF-κB signaling 28 , suggesting that increased NIK transcription in lead invading cells may facilitate mitochondrial energy dynamics that support collective invasion.
A hallmark of invasion is alteration of the extracellular matrix to facilitate cell mobility, which includes changes in the dynamics of such proteins as e-cadherins, integrins or matrix metalloproteinases. In conjugation to promoting invasion, NIK has also been shown to regulate matrix metalloproteinase 14 (MMP14) with a reduction in phosphorylated MMP14, or its active form, seen in cells lacking NIK, where the opposite held true in cells expressing a constitutively active form of NIK 12 . RNA-seq analysis also revealed elevated MMP9 expression in glioma cells stimulated with TWEAK, which has been associated with cancer biomarkers 18 . Furthermore, as collective invasion maintains cell-to-cell contact through the extracellular matrix, it has been shown that stabilization of integrins is associated with increased collective invasion of solid tumors 29 , coinciding with an increase in integrin alpha 11 and integrin beta 3 gene expression in highly invading, TWEAK-treated cells. Integrin alpha 11 and integrin beta 3 have also been linked to increased tumor progression in other cancer types [19][20][21] . RNA-seq disease pathway analysis also highlighted TWEAK-treated glioma cells as having higher activation of cell-cell junctions and tumor development, invasion, and metastasis. Elevated matrix metalloprotease, integrin, and tumor progression is consistent with a leader phenotype displayed by NIK-expressing cells during collective invasion.
NIK and NF-κB dysregulation has been highly correlated with the induction of disease and malignancies [30][31][32] . Several studies have demonstrated an increase in NIK expression in various cancer models. Of these studies, NIK elevation was observed in breast cancer, lymphomas, pancreatic cancer, gastric cancer, and GBMs 11,33-36 . Given the pro-tumorigenic role NIK has in cancer and our studies suggesting that upregulation of NIK gene expression by cytokines in the tumor microenvironment can robustly trigger invasion, the inhibition of NIK may prove a promising therapeutic target for primary as well as recurrent or invasive/metastatic tumors 22,37-39 .
Cell Lines BT25, BT114 and BT116 cell lines were obtained from human GBM patients as previously described 40 . These cell lines were maintained as spheroids in neural stem cell medium containing DMEM/F-12, 1x B-27 supplement minus vitamin A, 1x GlutaMAX, 25 ng/mL EGF, 25 ng/mL basic broblast growth factor (bFGF), and 1x penicillin/streptomycin (Life Technologies).
CRISPR-Cas9 gene knockout BT25, BT114, and BT116 cells were transduced with a mixture of LentiCrispR-v2 carrying three gRNAs for each target. The gRNA sequences for human NIK E2F4 and E2F5 are shown in Supplemental Table 1. RNA Sequencing 1 x 10 6 BT25 GBM cells harvested in untreated, TWEAK-treated (10 ng/mL for 4 hrs) and TNFα-treated (10 ng/mL for 30 minutes) conditions along with stable transgenic BT25 NIK KO cells. Duplicates of cell pellets were shipped on dry ice to Azenta by Genewiz (South Plain eld, NJ, USA), where RNA was isolated, and they conducted sequencing and constructed the sequencing library. For sequencing, HiSeq 2x150 bp was used. Sequence reads were trimmed using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome using the STAR aligner v.2.5.2b. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. The hit counts were summarized and reported using the gene_id feature in the annotation le. Only unique reads that fell within exon regions were counted. DESeq2 les were uploaded to Ingenuity Pathway Analysis software (IPA; QIAGEN, Germantown, MD, USA) for dataset comparisons and to analyze altered genes that were statistically signi cant compared to wild type. IPA software was also used to analyze overall changes in disease function pathways related to identi ed gene families. GraphPad Prism (San Diego, CA, USA) was used to generate volcano plots and heatmaps.

Immuno uorescence staining
Collagen-embedded spheroids were seeded on eight-well chamber slides (#80827, Ibidi, Munich, Germany) or 96 half-area well plates and allowed to adhere for 2 hours. During spheroid and monolayer invasion live cell imaging, cells were labeled with DiO or DiD for 30 minutes at 37°C before washing three times in media. Cells were allowed to invade for 48 to 72 hours, xed with 4% paraformaldehyde, and permeabilized for 20 minutes with 0.3% Triton X-100 in PBS. Cells were incubated overnight in 0.1% Triton X-100 and 1% BSA in PBS at 4°C. Cells were then incubated in 1% BSA for 1 hour at room temperature. Cells were counterstained with the nuclear stain DAPI (Invitrogen, P36931).

Three-dimensional collagen invasion assay
Monolayer invasion assays were performed as previously described [28]. Brie y, collagen type I (Corning, NY) was diluted to 2 mg/ml in DMEM/F-12 medium (1x Pen/Strep), and matrices were polymerized in 96well plates. A total of 4 x 105 cells cultured in NSCs or NSCs+10% serum were seeded in triplicate in 100 μl DMEM/F-12 (1x Pen/Strep, 1x Glutamax) without growth factors or serum. Cells were xed with 3% glutaraldehyde solution after 48 hours of invasion and stained with 0.1% toluidine blue. Invasion density was quanti ed by counting cells below the plane of the monolayer by bright-eld light microscopy using a 10 x 10 ocular grid at 10x or 20x magni cation corresponding to a 1 mm 2 eld. Numbers in at least three equivalent, random elds were counted (n = 3 wells each) and normalized to the corresponding control. All experiments were performed at least three times.
Live cell invasion assays were performed using GBM cells. The cells were collected and centrifuged at 1.0 rcf for 2.5 minutes, and the medium was removed and disassociated with accutase for 9 minutes at room temperature before centrifugation at 1.0 rcf for 2.5 minutes. Accutase was removed, and cells were resuspended in NSC media and then quanti ed. Approximately 2.0x10 6 cells were transferred into a 15 mL conical tube, and media was added to 2 mL with 12 µL of DiO added. Cells were incubated for 30 minutes at 37°C with DiO, followed by centrifugation at 1.0 rcf for 2 minutes. Cells were washed three times in NSC media before requanti cation. Cells were either used for monolayer invasion assay at 40,000 cells/well or 1.2x10 6 cells were incubated for one week at 37°C in NSC media before being embedded into 2.0 mg/ml collagen matrix.
After spheroid formation, spheres were collected at 1.0 rcf for 2.0 minutes and resuspended in 2 mL of fresh media. The collagen matrix was prepared, approximately 60 µL of resuspended spheres was added to the matrix, and 18 µL of spheres embedded in collagen was added to each well. Collagen was allowed to solidify for 2 hours at 37°C before taking initial images. Images were acquired over a 72-hour time course.

Image Acquisition
Images were acquired with a Nikon TI A1R inverted confocal microscope with a CFI60 Plan Apochromat Lambda 10x objective lens. Images were acquired with the following scan parameters: a "frame" scan mode of 1024 x 1024 pixels with a 16 bit depth and a grating of 3 rotations. Three-dimensional projections were obtained through Z stack images with 0.4700 µm between each image. RNA isolation, cDNA synthesis, and quantitative RT-qPCR Total RNA was isolated from cells by a Purelink™ RNA Mini Kit (Life Technologies). cDNA was synthesized from 1 μg of total RNA using iScript reverse transcription supermix (Bio-Rad, Hercules, CA) following the manufacturer's protocol. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) with a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The primers used are listed in Supplemental Table 2. The expression of mRNA was normalized to GAPDH expression levels. All experiments were performed at least three times with three replicates per sample.